Understanding the intracellular transport and localization of proteins is important because many processes in biology, including transcription, translation, and metabolic or signal transduction pathways, are mediated by proteins and non-covalently-associated multiprotein complexes localized to particular cellular compartments Reed, L. J. (1974) Multienzyme Complexes. Acc. Chem. Res. 7, 40-46. Proteins and protein complexes are the workhorses of the chemical machinery in living systems and define the interface between metabolic pathways, signaling pathways, and the response of cells to the environment. Since the completion of the genome sequencing projects, the focus of biological research has moved to identifying proteins involved in cellular processes, determining their functions and how, when, and where they localize to facilitate interaction with other proteins involved in specific pathways. Further, with rapid advances in genome sequencing projects there is a need to develop strategies to define “protein linkage maps”, detailed inventories of protein interactions that make up functional assemblies of proteins Lander, E. S. (1996) The new genomics—global views of biology. Science 274, 536-539; Evangelista, C., Lockshon, D. & Fields, S. (1996) The yeast two-hybrid system—prospects for protein linkage maps. Trends in Cell Biology 6, 196-199. Eukaryotic cells have specialized compartments containing specific proteins needed to carry out defined processes. Understanding when and where specific proteins localize is key to further understanding of cellular processes.
GFP and its numerous related fluorescent proteins are now in widespread use as protein tagging agents (for review, see Verkhusha et al., 2003, GFP-like fluorescent proteins and chromoproteins of the class Anthozoa. In: Protein Structures: Kaleidescope of Structural Properties and Functions, Ch. 18, pp. 405-439, Research Signpost, Kerala, India), and have been used to visualize protein trafficking in living cells (Llopis, McCaffery et al. 1998; Southward and Surette 2002). Generally, using existing methodologies, a gene of interest is expressed in fusion with GFP and the fusion protein localizes fluorescence to the normal localization of the target protein. The fusion protein may be encoded by a constitutive promoter on a plasmid or may be directly transfected into the cell by any transfection methodology (FIG. 1). A number of localization vectors expressing fluorescent proteins fused to subcellular localization sequences or tags have been described and/or are commercially available (e.g., the Living Colors™ localization vectors, BD Biosciences Clontech, Palo Alto, Calif.)
However, although protein localization methods using GFP fused to a protein of interest have been useful, these methods provide limited information and are compromised by problems inherent in using a relatively large terminally fused reporter protein tag. In particular, GFP and related fluorescent proteins may cause misfolding of the fused protein, and may interfere with protein processing and/or intracellular transport. Misfolding of the reporter can result in the generation of insoluble aggregates of the fusion, which may be unable to freely move through intracellular processing and transport systems within the cell. Additionally, GFP fusions may alter biophysical properties of a test protein, resulting in a default in the corresponding localization pathway (Hanson and Ziegler, 2004). In general, the use of GFP fusions in extracytoplasmic compartments has been limited because of export deficiencies. As an example, efforts to obtain functional GFP following export through the sec-dependent pathway failed because of improper folding of the protein; the secreted fraction of GFP was not fluorescent (Feilmeier, Iseminger et al. 2000; Tanudji, Hevi et al. 2002). Accordingly, it is difficult to obtain a true picture of a test protein's trafficking or distribution within a cell using terminal GFP fusion tags, and systems capable of visualizing subcellular compartmentalization through a wider lens and over time are needed.
GFP fragment reconstitution systems have been described, mainly for detecting protein-protein interactions, but none are capable of unassisted self-assembly into a correctly-folded, soluble and fluorescent re-constituted GFP, and no general split GFP folding reporter system has emerged from these approaches. For example, Ghosh et al, 2000, reported that two GFP fragments, corresponding to amino acids 1-157 and 158-238 of the GFP structure, could be reconstituted to yield a fluorescent product, in vitro or by coexpression in E. coli, when the individual fragments were fused to coiled-coil sequences capable of forming an antiparallel leucine zipper (Ghosh et al., 2000, Antiparallel leucine zipper-directed protein reassembly.: application to the green fluorescent protein. J. Am. Chem. Soc. 122: 5658-5659). Likewise, U.S. Pat. No. 6,780,599 describes the use of helical coils capable of forming anti-parallel leucine zippers to join split fragments of the GFP molecule. The patent specification establishes that reconstitution does not occur in the absence of complementary helical coils attached to the GFP fragments. In particular, the specification notes that control experiments in which GFP fragments without leucine zipper pairs “failed to show any green colonies, thus emphasizing the requirement for the presence of both NZ and CZ leucine zippers to mediate GFP assembly in vivo and in vitro.”
Similarly, Hu et al., 2002, showed that the interacting proteins bZIP and Rel, when fused to two fragments of GFP, can mediate GFP reconstitution by their interaction (Hu et al., 2002, Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9: 789-798). Nagai et al., 2001, showed that fragments of yellow fluorescent protein (YFP) fused to calmodulin and M13 could mediate the reconstitution of YFP in the presence of calcium (Nagai et al., 2001, Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98: 3197-3202). In a variation of this approach, Ozawa at al. fused calmodulin and M13 to two GFP fragments via self-splicing intein polypeptide sequences, thereby mediating the covalent reconstitution of the GFP fragments in the presence of calcium (Ozawa et al., 2001, A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal. Chem. 72: 5151-5157; Ozawa et al., 2002, Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem. 73: 5866-5874). One of these investigators subsequently reported application of this splicing-based GFP reconstitution system to cultured mammalian cells (Umezawa, 2003, Chem. Rec. 3: 22-28). More recently, Zhang et al., 2004, showed that the helical coil split GFP system of Ghosh et al., 2000, supra, could be used to reconstitute GFP (as well as YFP and CFP) fluorescence when coexpressed in C. elegans, and demonstrated the utility of this system in confirming coexpression in vivo (Zhang et al., 2004, Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119: 137-144).
Although the aforementioned GFP reconstitution systems provide advantages over the use of two spectrally distinct fluorescent protein tags, they are limited by the size of the fragments and correspondingly poor folding characteristics (Ghosh et al., Hu et al., supra), the requirement for a chemical ligation or fused interacting partner polypeptides to force reconstitution (Ghosh et al., 2000, supra; Ozawa et al., 2001, 2002 supra; Zhang et al., 2004, supra), and co-expression or co-refolding to produce detectable folded and fluorescent GFP (Ghosh et al., 2000; Hu et al., 2001, supra). Poor folding characteristics limit the use of these fragments to applications wherein the fragments are simultaneously expressed or simultaneously refolded together. Such fragments are not useful for in vitro assays requiring the long-term stability and solubility of the respective fragments prior to complementation. An example of an application for which such split protein fragments are not useful would be the quantification of polypeptides tagged with one member of the split protein pair, and subsequently detected by the addition of the complementary fragment.